This invention relates generally to the field of biopolymers, and more particularly relates to the purification of oligomers such as oligonucleotides, oligopeptides, oligosaccharides, and the like.
There is an increasing demand for oligonucleotides for use in nucleic acid hybridization assays, as polymerase chain reaction (xe2x80x9cPCRxe2x80x9d) primers or as sequencing primers in Sanger, or dideoxy, sequencing. Synthesis and purification of research-purpose quantities of oligonucleotides routinely yields product having purity of greater than 95%, but this high purity requires a lengthy, time-consuming, and labor intensive purification protocol. Typically, a 0.2 micromole-scale preparation requires a seven-step purification procedure: 1) preparing a purification gel; 2) loading the gel with the reaction mixture to be purified, iii) running the gel overnight; 4) visualizing and cutting the appropriate bands from the gel; 5) soaking the bands in elution buffer for two days to extract the desired product from the gel matrix; 6) manual desalting the extracted product on a reverse phase (xe2x80x9cRPxe2x80x9d) column and drying the solvent; and 7) manually precipitating the product from the solvent. The amount of the product obtained is quantitated using UV spectroscopy.
Any simplification of these lengthy, time consuming, and labor intensive purification protocols would be very valuable. Further, a purification scheme that could be automated and applied to oligomers other than oligonucleotides would be desirable as well.
During the process of oligonucleotide synthesis, depurinated sites can be introduced at random sites caused by prolonged exposure to acid; the final ammonium hydroxide deprotection step cleaves the oligonucleotide chain at the depurinated sites. McHugh et al. (1995) Nucleic Acids Research 23:1664-1670. Methods that were devised to simplify DNA purification by, e.g., Efcavitch et al. (1985) Nucleosides and Nucleotides 4:267 and McBride et al. (1988) BioTechniques 66:362-367, were only capable of purifying shorter DNA oligomers because they did not fully account for the complicating nature of the ammonium hydroxide cleavage products.
An enzymatic purification scheme has been reported in which an oligomer is first synthesized on a solid support. Urdea et al. (1986) Tetrahedron Lett. 27:2933-2936. Subsequent to preparation of the desired-length solid support-bound oligonucleotide, exocyclic amines and phosphate groups in the oligomer were deprotected without cleavage of the linkage to the support. The purification used spleen phosphodiesterase to digest failure sequences that did not contain a terminal 5xe2x80x2-benzoyl group of the full-length oligomeric product. The process resulted in oligomers of improved purity, but abasic sites in the product oligomer remained.
A rapid cartridge purification method has also been described by Horn et al. (1988) in Nucleic Acids Res. 16:11559-11571. The key step in this procedure is the cleavage of all apurinic sites in the oligomer with a solution of aqueous lysine prior to removal of the crude product from the solid support. As a result, essentially all of the truncated 5xe2x80x2xe2x80x94O-dimethoxytrityl (xe2x80x9cDMTxe2x80x9d)-containing oligomers are eliminated from the mixture of cleaved oligomers. The authors report that DNA oligomers of up to 118 bases in length were purified to near homogeneity using the procedure.
An approach related to that described in Horn et al. (1988) made use of a solid support with a disilyloxy linkage. Cleavage of abasic sites in the oligomer under very mild conditions, while the oligomer was still attached to the support, ensured that all 5xe2x80x2xe2x80x94O-DMT-containing molecules, when cleaved from the support, had correct 3xe2x80x2- and 5xe2x80x2-ends. Kwiatkowski et al. (1996) Nucleic Acids Res. 24:4632-4638.
Natt et al. (1997) Tetrahedron 53:9629-9636 describe an approach to oligomer purification that used a lipophilic capping reagent to cap failure sequences during synthesis. The lipophilic nature of the failure sequences made it possible to separate capped failure sequences from detritylated full-length oligomers chromatographically. However, the method was inefficient with respect to depurinated/cleaved sequences since the two families of species, i.e., the detritylated 5xe2x80x2 segment and the detritylated 3xe2x80x2 segment, do not contain the lipophilic capping group. The use of trityl groups with enhanced lipophilic properties as 5xe2x80x2xe2x80x94O protecting groups has been advocated to facilitate RP-high performance liquid chromatography (xe2x80x9cHPLCxe2x80x9d) purification. Ramage (1993) Tetrahedron Lett. 34:7133-7136. As with the approaches discussed above, this process is also limited with regard to cleaved abasic sites.
Purification approaches that involve a xe2x80x9ccapturexe2x80x9d step have been proposed. In each case, the 5xe2x80x2 end of the oligomer to be purified carries a moiety by which capture can be effected. For example, Bannwarth et al. (1990), in Helv. Chim. Acta 73: 1139-114, described a combined purification/phosphorylation procedure for oligodeoxynucleotides that included a capture step. A special ribonucleotide, N1xe2x80x94(MMTxe2x80x94Sxe2x80x94(CH2)10)xe2x80x942xe2x80x2,3xe2x80x2Bz2-rU-5xe2x80x2-xcex2-cyanoethyl (wherein xe2x80x9cMMTxe2x80x9d represents monomethoxytrityl and xe2x80x9cBzxe2x80x9d represents benzyl), containing a protected thiol and a diol system, was incorporated into the oligonucleotide during the final DNA synthesis cycle. After complete deprotection and removal of the MMTr protecting group, the oligomer with a 5xe2x80x2xe2x80x94SH group could be captured on a controlled pore glass (xe2x80x9cCPGxe2x80x9d) support having surface-bound-Sxe2x80x94S-pyridine groups; contaminating oligomers were removed by washing. The purified oligomer was released from the capture support after oxidative cleavage of the ribo-diol system and beta-elimination under basic conditions.
A purification procedure using a photolabile 5xe2x80x2-biotin reagent to capture oligomers on a avidin capture support has also been described. Olejnik et al. (1996) Nucleic Acids Research 24:361-366. The linking groups could be cleaved by photolysis to release the product oligomer in the 5xe2x80x2-phosphate form.
Synthesis and purification of 5xe2x80x2-mercaptoalkylated oligonucleotides has been described in which thiolated oligomers were purified by a single-step covalent chromatography procedure using an activated sulfhydryl support. Kumar et al. (1996) Bioorg. Med. Chem. Lett. 6:683-688.
In addition, purification of proteins by taking advantage of the selectivity of unique nickel-nitrilotriacetic acid (xe2x80x9cNixe2x80x94NTAxe2x80x9d) solid supports with an affinity tag consisting of six consecutive histidine residues has been known for years. This type of immobilized metal affinity chromatography (xe2x80x9cIMACxe2x80x9d) has been used for sequence-specific isolation of nucleic acids by peptide nucleic acids (xe2x80x9cPNAxe2x80x9d)-controlled hybrid selection using oligohistidine-PNA chimera (the chemistry of PNA and peptide assembly are essentially identical). Orum et al. (1995) BioTechniques 19:472-480. The system has been extended to synthetic DNA oligomers containing six consecutive 6-histaminylpurine (xe2x80x9cHisxe2x80x9d) nucleotides, introduced using a convertible nucleotide phosphoramidite and further derivatized to form the His nucleotides. Min et al (1996) Nucleic Acids Research 24:3806-3810. The His6-tagged strand was selectively retained by a Nixe2x80x94NTA-agarose chromatography matrix and the captured DNA thereafter eluted from the resin.
Background references that relate generally to methods for synthesizing oligonucleotides include those related to 5xe2x80x2- to -3xe2x80x2 syntheses based on the use of xcex2-cyanoethyl phosphate protecting groups, e.g., de Napoli et al. (1984) Gazz. Chim. Ital. 114:65, Rosenthal et al. (1983) Tetrahedron Lett. 24:1691, Belagaje et al. (1977) Nucl. Acids Res. 10:6295, and those references that describe solution-phase 5xe2x80x2- to -3xe2x80x2 syntheses, such as Hayatsu et al. (1957) J. Am. Chem. Soc. 89:3880, Gait et al. (1977) Nucl. Acids Res. 4:1135, Cramer et al. (1968) Angew. Chem. Int. Ed. Engl. 7:473, and Blackburn et al. (1967) J. Chem. Soc. Part C, 2438.
In addition to the above-cited art, Matteucci et al. (1981) J. Am. Chem. Soc. 103:3185-3191, describes the use of phosphochloridites in the preparation of oligonucleotides. Beaucage et al. (1981) Tetrahedron Lett. 22:1859-1862, and U.S. Pat. No. 4,415,732 describe the use of phosphoramidites in the preparation of oligonucleotides. Smith (1983) ABL 15-24, the references cited therein and Warner et al. (1984) DNA 3:401-411 describe automated solid-phase oligodeoxyribonucleotide synthesis.
U.S. Pat. Nos. 4,483,964 and 4,517,338 to Urdea et al. describe a method for synthesizing polynucleotides by selectively introducing reagents to a solid phase substrate in a tubular reaction zone. U.S. Pat. No. 4,910,300 to Horn et al. also describes a method for synthesizing oligonucleotides by sequentially adding nucleotidic monomers to a growing chain, but involves the incorporation of labeled, N4-modified cytosine residues at predetermined, spaced apart positions. U.S. Pat. No. 5,256,549 to Horn et al. describes a method for preparing oligonucleotides that involves a combination technique, i.e., in which the desired oligonucleotide is essentially synthesized and xe2x80x9cpurifiedxe2x80x9d simultaneously, such that the final product is produced in substantially pure form.
Horn et al. (1986) DNA 5(5):421-425 describes phosphorylation of solid-supported DNA fragments using bis(cyanoethoxy)-N,N-diisopropyl-aminophosphine. See also, Horn et al. (1986) Tetrahedron Lett. 27:4705-4708.
Horne et al. (1990) J. Am. Chem. Soc. 112:2435-2437 and Froehler et al. (1992) Biochemistry 31:1603-1609 relate to oligonucleotide-directed triple helix formation.
U.S. Pat. Nos. 5,594,117 and 5,430,136 to Urdea et al. disclose methods and reagents, e.g., modified monomeric reagents, for synthesizing oligonucleotides containing abasic, selectably cleavable sites. Oligonucleotides prepared having such sites are selectably cleavable by photolysis or by chemical or enzymatic reagents, e.g., reducing agents.
Methods for production of oligosaccharides are known as well. For example, Kanie et al. (1992) Curr. Opin. Struct. Biol. 2:674-681 and Ding et al. (1995) Adv. Exp. Med. Biol. 376:261-269 describe chemical synthesis of oligosaccharides. In order to synthesize saccharide oligomers of defined structure, orthogonal protecting groups are provided on the hydroxyl moieties of the monosaccharides that are sequentially added to the growing oligosaccharide chain. Acetyl and benzyl protecting groups are commonly used. A saccharide moiety may become an acceptor and thus able to combine with another saccharide by replacing a hydroxyl hydrogen with, for example, p-s-xcfx86xe2x80x94CH3 (p-methylphenylthio), xe2x80x94(CH2)nCOOCH3 or xe2x80x94(CH2)nxe2x80x94Oxe2x80x94xcfx86xe2x80x94OCH3. U.S. Pat. No. 4,701,494 to Graafland is also of interest as a process for the preparation of water soluble vinyl saccharide polymers is disclosed.
Accordingly, it is evident that many procedures have been developed for producing oligomers of nucleotides, amino acids, saccharides, and other monomers. These procedures for the most part rely on attaching a first monomer. Each subsequent monomeric unit is then added sequentially, with each addition involving a number of chemical reactions.
At each stage during the synthesis of the oligomer, there is a small but finite probability that a number of chains may not have been extended. Therefore, during the entire oligomerization process, a large number of errors may be introduced. These erroneous sequences (or xe2x80x9cfailure sequencesxe2x80x9d) that may manifest themselves in a number of ways. Without an adequate purification process to remove failure sequences, the error may lead to undesired products, suboptimum performance, and the like.
It has therefore become of increasing importance to be able to prepare oligomers with an assurance that there is substantially no contamination with oligomers having sequences that approximate but differ from the desired sequence. By removing failure sequences at the outset, one may avoid the need for subsequent purification steps, such as electrophoresis, which can result in loss of material; loss of material can of course be a serious problem when working with very small quantities of materials.
Accordingly, it is a primary object of the invention to address the aforementioned need in the art by providing a simplified, efficient and versatile method for purifying oligomers.
It is another object of the invention to provide a such a method wherein the oligomer is an oligonucleotide, an oligopeptide, an oligosaccharide, or the like.
It is an additional object of the invention to provide such a method wherein a support-bound oligomer is purified using alternating cleavage and capture steps.
It is yet another object of the invention to provide a method of purifying a synthetic oligonucleotide by performing both a 5xe2x80x2-selection step and a 3xe2x80x2-selection step.
In one aspect of the invention, then, a method is provided for preparing an oligomer segment of interest in purified form. Initially, the method involves sequentially coupling monomers to the terminus of a growing support-bound oligomer chain until the desired support-bound oligomer is obtained. The support-bound oligomer contains a first selectably cleavable linkage, a second selectably cleavable linkage, and a third selectably cleavable linkage, wherein the oligomer segment of interest is the segment flanked by the second and third selectably cleavable linkages, and wherein a first capture moiety is present at the free terminus of the support-bound oligomer, and a second capture moiety is present between the first and third selectably cleavable linkages. The selectably cleavable linkages and the capture moieties are introduced during synthesis using techniques described herein and/or known to those of ordinary skill in the art. Following synthesis of the support-bound oligomer, the following steps are carried out to provide the oligomer segment of interest in purified form: (a) the first selectably cleavable linkage is cleaved so as to release the oligomeric product from the solid support; (b) the released oligomeric product is incubated with a first capture medium which couples to the first capture moiety, and the xe2x80x9ccapturedxe2x80x9d oligomeric product is then isolated and optionally purified; (c) the second selectably cleavable linkage is cleaved to produce the oligomer segment of interest terminating in the second capture moiety; (d) the oligomer segment provided in step (c) is incubated with a second capture medium which couples to the second capture moiety, and the captured oligomer segment is then isolated and optionally purified; and (e) the third selectably cleavable linkage is cleaved to give the oligomer segment of interest in purified form.
Another aspect of the invention relates to the support-bound oligomeric product synthesized as just described and useful as a starting material in providing the purified oligomer segment of interest. The support-bound oligomeric product has the structural formula (I)
Sxe2x80x94[X1]n1xe2x80x94SC1xe2x80x94CP2xe2x80x94[X2]n2xe2x80x94SC3xe2x80x94T1xe2x80x94Xxe2x80x94T2xe2x80x94SC2xe2x80x94CP1xe2x80x83xe2x80x83(I)
wherein S represents the solid support, X1 and X2 are monomers or oligomeric segments, n1 and n2 are independently zero or 1, SC1, SC2 and SC3 represent first, second and third selectably cleavable sites, CP1 and CP2 represent first and second capture moieties, T1 is the first terminus of the oligomer segment of interest X, and T2 is the second terminus of the oligomer segment X. The support-bound oligomeric product of formula (I) may be used in the process described above to provide the oligomeric segment of interest X, terminating in T1 and T2, in purified form.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention.
For the sake of clarity, and without intent to limit the invention to any particular embodiment, the following discussion of the invention is related to the purification of an oligomer that is an oligonucleotide. When the oligomer is an oligonucleotide, T2 and T1 represent the 5xe2x80x2 and 3xe2x80x2 termini, respectively, SC2 represents a 5xe2x80x2-cleavable linkage, and SC3 represents a 3xe2x80x2-cleavable linkage. Those of ordinary skill in the art will recognize that, with minor modification, the methods disclosed and claimed herein can be applied to the purification of other oligomers, as well, e.g., oligopeptides, oligosaccharides and the like.
An oligonucleotide is provided having the structure of formula (I) wherein X is an oligonucleotide segment of interest and X1 and X2 are individual nucleotides or oligonucleotide segments. Purification of the oligonucleotide segment of interest is effected by cleavage at SC 1, incubation of the released product CP2xe2x80x94[X2]n2xe2x80x94SC3xe2x80x94T1xe2x80x94Xxe2x80x94T2xe2x80x94SC2xe2x80x94CP1 with a first capture medium CM1 comprised of a reverse phase chromatography or hydrophobic interaction chromatography medium, cleavage at SC2, incubation of the resulting product CP2xe2x80x94[X2]n2xe2x80x94SC3xe2x80x94T1xe2x80x94Xxe2x80x94T2 with a second capture medium CM2 comprised of a reverse phase chromatography or hydrophobic interaction chromatography medium, and cleavage at SC3 to give the purified oligonucleotide segment of interest T1xe2x80x94Xxe2x80x94T2.